Abstract
The plasma membrane nanoscale distribution of H-ras is regulated by guanine nucleotide binding. To explore the structural basis of H-ras membrane organization, we combined molecular dynamic simulations and medium-throughput FRET measurements on live cells. We extracted a set of FRET values, termed a FRET vector, to describe the lateral segregation and orientation of H-ras with respect to a large set of nanodomain markers. We show that mutation of basic residues in helix alpha 4 or the hypervariable region (HVR) selectively alter the FRET vectors of GTP- or GDP-loaded H-ras, demonstrating a critical role for these residues in stabilizing GTP- or GDP-H-ras interactions with the plasma membrane. By a similar analysis, we find that the beta 2-beta 3 loop and helix alpha 5 are involved in a novel conformational switch that operates through helix alpha 4 and the HVR to reorient the H-ras G-domain with respect to the plasma membrane. Perturbation of these switch elements enhances MAPK activation by stabilizing GTP-H-ras in a more productive signalling conformation. The results illustrate how the plasma membrane spatially constrains signalling conformations by acting as a semi-neutral interaction partner.
Original language | Undefined/Unknown |
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Pages (from-to) | 727–735 |
Number of pages | 9 |
Journal | EMBO Journal |
Volume | 27 |
Issue number | 5 |
DOIs | |
Publication status | Published - 2008 |
MoE publication type | A1 Journal article-refereed |
Keywords
- fRET
- microdomain
- nanocluster
- plasma membrane
- Ras